This small grant application is submitted to support preliminary studies on the mechanism of phosphorylation of the serotonin 5-HT1A receptor by protein kinase C. The principal investigator is a new investigator in the field of receptor biochemistry and will incorporate peptide mass spectrometric methods to identify the putative protein kinase C phosphorylation sites on the i3 loop of the receptor. The long-term goal of this grant is to establish methods that will allow us to develop projects to investigate the phosphorylation sites on the intact membrane bound receptor 5-Hydroxytryptamine (serotonin, 5-HT) is a biogenic amine neurotransmitter first discovered as a potent vasotonic factor in 1948. The cellular and physiological effects of serotonin are mediated by seven major families of receptors (5-HTn, where n= 1-7), containing at least 14 subtypes. The first 5-HT receptor cloned and by far the best-characterized 5-HT receptor is the 5-HT1A receptor. The 5-HT1A receptor has been shown to be important in a number of neurochemically related physiological processes especially those involving memory, depression, aggression, and anxiety. The 5-HT receptors are G-protein coupled receptors (GPCRs). GPCRs are characterized by their signaling via G proteins (guanine nucleotide binding proteins) upon activation. Desensitization of a receptor can occur after prolonged exposure to a stimulus such as a hormone or neurotransmitter disrupting the G protein signal pathway. One of the cellular events implicated in receptor desensitization is the kinase-directed phosphorylation of the associated receptor. The overall goal of this study is to identify some of the phosphorylation sites on the serotonin receptor 1A (5- HT1A) through the use of peptide mass spectrometry. Initial studies will focus on phosphorylation of purified peptides representing the third putative intracellular loop of the receptor and will involve development of mass spectrometric techniques for unambiguous identification of the sites of phosphorylation. The specific aims of this proposal are: (1) to identify, express, and isolate a peptide that mimics the third intracellular loop of the 5-HTIA receptor (5-HTla-i3), an approximately 135 amino acid region from R216 to M350 as a target for PKC; (2) to develop specific conditions under which either purified PKC or activated PKC from stimulated cell lysates can phosphorylate the 5-HT1A-i3 loop, and (3) to identify the specific phosphorylation sites on 5-HT1A-i3 by MALDI and HPLC-ESI mass spectrometry.